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The efflux transporter P-glycoprotein (P-gp) is an important component of the blood-brain barrier (BBB) that limits accumulation of many compounds in brain. Some opioids have been shown to interact with P-gp in vitro and in vivo. Genetic or chemical disruption of P-gp has been shown to enhance the antinociceptive and/or toxic effects of some opioids, although the extent of this phenomenon has yet to be understood. The purpose of this study was to assess quantitatively the influence of mdr1a P-gp on initial brain uptake of chemically diverse opioids in mice. The brain uptake of opioids selective for the mu (fentanyl, loperamide, meperidine, methadone, and morphine), delta (deltorphin II, DPDPE, naltrindole, SNC 121) and kappa (bremazocine and U-69593) receptor subtypes was determined in P-gp-competent (wild-type) and P-gp-deficient [mdr1a(-/-)] mice with an in situ brain perfusion model. BBB permeability of the opioids varied by several orders of magnitude in both mouse strains. The difference in brain uptake between P-gp-competent and P-gp-deficient mice ranged from no detectable effect (meperidine) to >/=8-fold increase in uptake (DPDPE, loperamide, and SNC 121). In addition, loperamide efflux at the BBB was inhibited by quinidine. These results demonstrate that P-gp modulation of opioid brain uptake varies substantially within this class of compounds, regardless of receptor subtype. P-gp-mediated efflux of opioids at the BBB may influence the onset, magnitude, and duration of analgesic response. The variable influence of P-gp on opioid brain distribution may be an important issue in the context of pharmacologic pain control and drug interactions.

Many opioids are substrates of the efflux transporter P-glycoprotein (P-gp) in the blood-brain barrier (BBB). In situ brain perfusion in wild-type and mdr 1a(-/-) P-gp-deficient mice was utilized to investigate potential P-gp-mediated transport of novel nonpeptidic delta agonists (AR-M delta compounds). Because radioactive compounds were not available for this series, liquid chromatography-mass spectrometric detection (LC-MS) was the assay methodology of choice. Verapamil in the perfusion buffer (0.5 microM) served as a positive control for P-gp-mediated efflux and as an experimental internal standard for P-gp modulation by AR-M delta compounds. LC-MS provided excellent assay sensitivity with no significant interferences. In P-gp-competent mice, the brain extraction of AR-M delta compounds ranged from 1.1 to 96%. The ratio of initial brain uptake clearances (Cl(up)) in P-gp-deficient and wild-type mice (P-gp effect) ranged from 0.96 to 4.91. Some compounds increased the Cl(up) of verapamil in P-gp-competent mice, consistent with P-gp inhibition. These results demonstrate that LC-MS is an appropriate assay methodology for mouse brain perfusion samples, that AR-M delta compounds may interact with P-gp in the BBB, and that the internal strategy can provide useful information concerning P-gp modulation by compounds of interest., (Copyright 2002 Wiley-Liss, Inc.)

Purpose: This study assessed the influence of mdr1a P-glycoprotein (P-gp) gene disruption, gender and concentration on initial brain uptake clearance (Clup) of morphine, quinidine and verapamil., Methods: Clup of radiolabeled substrates was determined in P-gp-competent and deficient [mdr1a(-/-)] mice by in situ brain perfusion. Brain:plasma distribution of substrates after i.v. administration was determined in both strains., Results: Genetic disruption of mdr1a P-gp resulted in 1.3-, 6.6- and 14-fold increases in Clup for morphine, verapamil and quinidine, respectively. With the exception of small differences for verapamil, gender did not affect Clup. Saturable transport of verapamil and quinidine was observed only in P-gp-competent mice, with apparent IC50 values for efflux of 8.6 +/- 2.3 microM and 36 +/- 2 microM, respectively. Verapamil Clup was approximately 50% higher in mdr1a(+/-) vs. mdr1a(+/+) mice; no such difference was observed for quinidine. In P-gp-competent mice, uptake of verapamil and quinidine was unaffected by organic vehicles. Plasma decreased VER Clup to a greater extent in the presence of P-gp. The influence of P-gp in situ was lower than, but correlated with, the effect in vivo., Conclusions: P-gp decreases Clup of morphine, verapamil and quinidine in situ with little or no influence of gender, but this effect cannot fully account for the effects of P-gp in vivo. P-gp is the only saturable transport mechanism for verapamil and quinidine at the murine blood-brain barrier. The influence of protein binding on Clup may be enhanced by P-gp-mediated efflux.

Purpose: This study was conducted to assess the influence of P-glycoprotein (P-gp) on brain uptake of multidrug resistance sensitive drugs using an in situ brain perfusion technique in P-gp-deficient (mdr1a[-/-]) and wild-type mice., Methods: The blood-brain transport of radiolabeled vinblastine, vincristine, doxorubicin, colchicine, and morphine was evaluated in mdr1a(-/-) and wild-type CF-1 mice with the in situ brain perfusion technique. Brain uptake of drugs after intravenous pretreatment with P-gp reversal agents, (PSC 833, GF 120918, or (+/-)-verapamil), or vehicle also was studied in wild-type mice. In all experiments, cerebral vascular volume was determined by co-perfusion of sucrose., Results: Cerebral vascular volume was preserved during perfusion, indicating maintenance of blood-brain barrier integrity in both types of mice within the concentration range of substrates in the perfusate. The apparent brain transport of colchicine. vinblastine, doxorubicin, and morphine was increased 3.0, 2.7, 1.5, and 1.4-fold, respectively, in mdr1a(-/-) mice compared with the wild-type: the brain uptake of vincristine was not affected by P-gp. Preadministration of PSC 833 or GF 120918 in wild-type mice led to a -3-fold increase in the brain transport of colchicine and vinblastine, but no effect was observed for the other compounds. Intravenous verapamil enhanced colchicine brain transport (1.8-fold), but failed to increase the brain uptake of vinblastine and morphine., Conclusion: The in situ brain perfusion technique appears to be a sensitive and powerful tool for medium throughput screening of the brain uptake of multidrug resistance sensitive drugs. The effect of P-gp is characterized more efficiently with mdr1a(-/-) mice than by using modulators of P-gp in wild-type mice.

An in situ mouse brain perfusion model predictive of passive and carrier-mediated transport across the blood-brain barrier (BBB) was developed and applied to mdr1a P-glycoprotein (Pgp)-deficient mice [mdr1a(-/-)]. Cerebral flow was estimated from diazepam uptake. Physical integrity of the BBB was assessed with sucrose/inulin spaces; functional integrity was assessed with glucose uptake, which was saturable with a Km of approximately 17 mmol/L and Vmax of 310 mmol x 100 g(-1) x min(-1). Brain uptake of a Pgp substrate (colchicine) was significantly enhanced (two- to fourfold) in mdr1a(-/-) mice. These data suggest that the model is applicable to elucidating the effects of efflux transporters, including Pgp, on brain uptake.